The present invention relates to pharmaceutical compositions in form of solid lipidic nanospheres able to fast penetrate into the cells, the process for their preparation and their use in the treatment of tumoral pathologies and of Mediterranean anaemia.
It is known from literature that the carboxylic acids having a low number of carbon atoms, such as acetic acid, propionic acid, butyric acid and succinic acid, and their derivatives, may inhibit the proliferation of the tumoral cells, in particular in the case of colon cancers (H. P. Scheppach, F. Ritcher, Eur. J. Cancer Prevention, 4, 373-378, 1995, and 31, 1077-1080, 1995).
There are in particular experimental proofs showing the antiproliferative activity of the butyric acid salts, for example of the sodium salt, towards a great variety of neoplastic cells (S. P. Landon et al., Cancer res., 48, 6161-6165, 1988; D. Coradini et al., Cell Prolif., 30, 149-159, 1997; H. Yamamoto et al., Int. J. Cancer, 76, 897-902, 1998). Recent studies showed that sodium butyrate is able to modulate the expression of the oncogenes and of the genes regulating the apoptosis in cells from different histotypes (O. C. Velasquez et al., J. Parenteral Enteral Nutr., 20, 243-250, 1996; M. Mandal, R. Kumar, Cell Growth Diff., 7, 311-318, 1996).
It is moreover known that the carboxylic acids themselves andlor certain derivatives thereof may help in a significant way, in the case of the Mediterranean anaemia, the transformation of xcex2-globin to xcex3-globin, or fetal globin, resulting in an improvement of the disease (S. P. Perine et al., New England J. Medicine, 328, 81-86, 1993, A. F. Collins et al., Blood, 85, 43-49, 1995).
At present, the use of such compounds is however strongly limited by the difficulty in reaching effective plasmatic concentrations, owing to the short half-life time, which makes the metabolism and the excretion of said substances too fast. In order to obtain satisfactory results therefore one ought to administrate high amounts of acid, with the drawback of causing harmful side effects.
Therefore one feels the need to have an adequate system of release for these substances, which allows to decrease their doses, thus minimising the side effects.
The document WO 94/20072 (Westesen K. at al.) describes particles of bioactive agents wherein the matrix is constituted by the bioactive agent itself. Substances particularly suitable for the formulation as said particles are drugs and other bioactive materials which are poorly water soluble. A long list of said substances, from anesthetics to virustatics, comprising tocopherol acetate and tocopherol succinate is reported.
No information about the internalization of said particles into cells is given.
Now the Applicant found new pharmaceutical compositions allowing to overcome the drawbacks of the prior art, showing a surprisingly high biological activity.
Said pharmaceutical compositions are prepared in form of solid lipidic nanospheres characterised in that they comprise as an active substance a lipidic substance consisting of cholesteryl butyrate and if necessary one or more further pharmacologically active substances.
A further object of the present invention is the process for the preparation of said lipidic nanospheres, comprising the following steps:
a) heating of a mixture comprising a lipidic substance and one or more surfactants at a temperature such as to take the mixture to melting;
b) heating of a mixture consisting of water and one or more co-surfactants at a temperature at least equal to the step a) one;
c) hot mixing under mild stirring of the mixture of the step b) with the mixture of the step a), with the achievement of a microemulsion;
d) dispersion of the microemulsion obtained in the step c) in pre-cooled water;
e) washing of the dispersion of the step d) with distilled water by diafiltration;
f) freeze-drying of the product obtained in the step e) or its hot sterilisation, characterised in that said lipidic substance consists of cholesteryl butyrate.
The pharmaceutical compositions in form of solid lipidic nanospheres object of the present invention are useful in the treatment of all the pathological conditions for which the administration of the above mentioned carboxylic acids is effective, and they are particularly suitable for the treatment of tumoral pathologies and of the Mediterranean anaemia.
The characteristics and the advantages of the solid lipidic nanospheres as a release system for the carboxylic acids according to the present invention and of the related preparation process will be pointed out in detail in the following description.
The present invention relates to pharmaceutical compositions in form of solid lipidic nanospheres obtained from microemulsions of a lipidic substance, stabilised by at least a surfactant and by one or more cosurfactants.
With the term lipidic nanospheres in the present invention we mean particles having an average diameter lower than 300 nm.
For the preparation of said microemulsions a lipidic substance in a mixture with one or more surfactants, and if necessary one or more further pharmacologically active substances, is heated to melting; separately a mixture consisting of water and one or more cosurfactants is heated to a temperature at least equal to that one at which the mixture containing the lipidic substance melts. The aqueous mixture is then hot added under mild stirring to the mixture containing the lipidic substance, obtaining a microemulsion.
The so obtained microemulsion is poured into precooled water at a temperature ranging from 2 to 10xc2x0 C. under mild stirring, using a water amount ranging from 10:1 to 80:1 parts by volume with respect to the volume of the microemulsion. The so obtained dispersion is then washed many times with distilled water by diafiltration in order to remove the components soluble in water, using a TCF2 equipment (Amicon-Grace-Danvers, USA) equipped with a YM 100 Diaflo membrane with a 100,000 Dalton cut-off, as disclosed in R. Cavalli et al., S. T. P. Pharma Sciences, 2(6), 514-518, 1992.
Such dispersion is finally hot sterilised in autoclave at 121xc2x0 C. for 15 minutes at 2 atm, or freeze-dried.
The so obtained lipidic nanospheres have an average diameter ranging from 40 to 300 nm, and preferably from 100 to 200 nm, and a polydispersion index ranging from 0.10 to 0.50.
The characterisation of the microemulsions has been carried out by photocorrelation spectroscopy with a N 4 Coulter instrument, as disclosed in R. Cavalli et al., Int. J. Pharm., 148, 47-54, 1997.
Said lipidic substance constitutes the essential active substance of the lipidic nanospheres, which however may include, in particular embodiment forms of the present invention, one or more other pharmacologically active substances.
Such further active substances are typically selected from the group consisting of doxorubicin, idarubicin and taxol.
According to a preferred embodiment form of the present invention said lipidic substance is cholesteryl butyrate.
As surfactants phosphatidylcholine taken from soy or egg yolk, phospholipids and their mixtures are typically used.
According to a preferred embodiment form of the present invention the used surfactant is a commercial product known with the name Epikuron 200(copyright) (Lukas Meyer, Hamburg, Germany), consisting of phosphatidylcholine for 95%.
The cosurfactants are selected from alcohols, such as butyl alcohol, carboxylic acids such as butyric and hexanoic acid and bile salts such as sodium taurocholate and sodium glycocholate.
According to a preferred embodiment form of the present invention, in the preparation of the microemulsion the various substances are used in the following proportions, expressed as percentages by weight with respect to the total weight of the microemulsion:
In the preferred embodiment form the lipidic nanospheres obtained by diafiltration have a titre in lipidic substance ranging from 25 to 42%, the residue consisting of phosphatidylcholine and/or phospholipids and by traces of other substances used in the preparation process.
According to a particularly preferred embodiment form of the present invention the composition of the solid lipidic nanospheres, expressed as percentage by weight of the various components, is the following one:
Owing to their small size and their composition, said nanospheres have the unexpected characteristic to be rapidly internalised into cells, where the active substance is fast released.
With respect to the systems of the prior art, said solid lipidic nanospheres therefore are the ideal release system for active substances such as the low molecular weight carboxylic acids present in the esters of the present invention. In fact they allow a strong reduction of the effective doses, with subsequent limitation of the side effects.
The pharmaceutical compositions in form of solid lipidic nanospheres of the present invention may therefore be successfully used in the treatment of all the pathologies for which the above mentioned characteristics of fast internalisation of the active substance into the cells are important.
The pharmaceutical compositions in form of lipidic nanospheres object of the present invention are useful in the treatment of all the pathologies for which the administration of acetic acid, propionic acid, butyric acid and succinic acid is effective, and in particular in the treatment of the tumoral pathologies and of Mediterranean anaemia.
For said uses the nanospheres according to the invention may be used alone, or in a mixture with pharmacologically acceptable excipients and/or diluents, and/or pharmacologically active substances. In particular embodiment forms of the pharmaceutical compositions according to the present invention, said active substances are antineoplastic agents.
The following examples of preparation of pharmaceutical compositions in form of solid lipidic nanospheres are reported for illustrative, but not limitative purpose of the present invention.